Insights into HuR RRM1–2 tandem domains self‐association and mRNA recognition

Abstract

Human antigen R (HuR) employs its mRNA stabilizing activity to function as a major post‐transcriptional regulator of gene expression. This protein plays a central role in the outcome of the inflammatory response as it may stabilize mRNAs of key components of the immune system. HuR comprises three functionally distinct RNA Recognition Motifs: RRM1, RRM2, and RRM3. The two N‐terminal tandem RRM domains can selectively bind AU‐rich elements (AREs), while the third RRM domain (RRM3) contributes to interactions with the poly‐A tail of target mRNA. Here, we characterized the structure, dynamics, and binding properties of HuR RRM11‐99 and RRM1–21‐189 tandem domains using a combination of Nuclear Magnetic Resonance and ion mobility‐mass spectrometry. RRM11‐99 adopts a typical RRM fold, composed of two α‐helices and a 4‐stranded β‐sheet (β1α1β2β3α2β4 topology and a β‐hairpin between α2 and β4). 15N relaxation measurements demonstrate that the N‐terminal intrinsically disordered region (residues 1–19) and the RRM1‐RRM2 linker region (residues 95–106) are highly flexible, suggesting that RRM1 and RRM2 domains behave as independent structural modules in solution. NMR titration experiments show that residues on the β‐sheet surface (β1 and β3) and the C‐terminus of RRM11‐99 mediate mRNA binding. An increase in R2/R1 values further confirms the mRNA interaction site, which is compatible with a low μM binding affinity. Both the single RRM11‐99 and the tandem RRM1–21‐189 have the ability to self‐associate, although representing a minor population. Ion mobility‐mass spectrometry identified the presence of oligomers (dimers, trimers and tetramers) with at least three different conformations for the apo forms of RRM11‐99 and RRM1–21‐189. However, only the monomeric species was able to interact with RNA, suggesting that ligand binding shifts the oligomeric/conformational equilibrium of HuR N‐terminal RRM domains. Our results show that the tandem RRM1–2 domains of HuR adopt three distinct conformations in the absence of RNA, suggesting that conformational selection may underlie the mechanism by which HuR recognizes and stabilizes mRNA.Support or Funding InformationThis work was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and by a Brazil Initiative Collaboration Grant from Brown University to A.S.P.